Listeria monocytogenes penetrates and multiplies within professional phagocytes and other cells such as the Caco-2 human enterocytelike cell line. Listeriolysin O, a membrane-damaging cytotoxin accounts for intracellular multiplication through lysis of the membrane-bound phagocytic vacuole. This work demonstrates that once released within the cytosol, L. monocytogenes acquires the capacity to spread intracellularly and infect adjacent cells by interacting with host cell microfilaments. Such evidence was obtained by using drugs which disrupt the cell cytoskeleton. Nocodazole, which blocks polymerization of microtubules, did not affect intracellular spread, whereas cytochalasin D, which blocks polymerization of G-actin, inhibited the intracellular motility of the bacteria. By using fluorescence staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), transmission electron microscopy, and immunogold labeling, direct evidence was obtained that intracellular bacteria were enveloped with a thick layer of F-actin. Within 2 h after entry, it was demonstrated by confocal microscopy that bacteria were following highly organized routes corresponding to stress fibers. Four hours after entry, some bacteria presented random movements which could be seen by the presence of a large trail of F-actin. Such movements also caused protrusions which deeply penetrated adjacent cells and resulted in the formation of vacuoles limited by a double membrane. After subsequent lysis of these membranes, bacteria released within the cytoplasm were able to multiply and invade new cells. In contrast, an hly::Tn1545 mutant of the wild-type microorganism demonstrated almost no intracellular spread. Only a few bacteria displaying delayed lysis of the phagocytic vacuole behaved like the wild-type strain. Hemolysin-mediated lysis of the phagocytic vacuole and subsequent interaction with host cell microfilaments may represent a major virulence factor allowing tissue colonization during listeriosis.